Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)

Put 60 µl of your sample in tube S1

Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2

Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3

Prepare your Microplate

Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well

Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds

Incubate plate at 37C for 30 minutes

Remove plate and measure the absorbance at 562 nm on a plate reader

Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown

Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl. Use the standard curve to determine the protein concentration of each unknown sample